[unreadable] The long-term objective of this project is to develop multi-modal (MRI, fluorescence) 129Xe biosensors for early cancer detection, and possible therapeutic intervention. Cryptophanes bind xenon more tightly within their interior (Kd ~-10O [unreadable]m) than other organic molecules, making these cages attractive for xenon imaging experiments. Xenon gas is sensitive to its molecular environment, and small perturbations near the cryptophane produce large changes in the 129Xe NMR chemical shift. Moreover, xenon is biologically compatible, diffuses readily in vivo, and can be "hyperpolarized" to enhance the NMR signal 10000-fold. These properties form the basis for a sensitive imaging reagent capable of simultaneously monitoring multiple analytes in vivo, each causing a unique 129Xe chemical shift. This "multiplexing" capability offers unparalleled opportunities for molecular imaging of tumorigenesis, where simultaneous identification of multiple cancer markers would speed diagnosis, and improve treatment. Two particularly challenging, but important goals are the early detection of pancreatic and brain cancers, as these neoplasms are a leading cause of cancer death in the U.S., and their regulation is poorly understood at the molecular level. [unreadable] In the P21 portion of this proposal, Aim #1 (year 1) is to synthesize cryptophanes and other carriers that will have greater affinity and capacity for xenon, longer 129Xe hyperpolarized spin-lattice relaxation times, and different chemical shifts. 29Xe*cryptophane binding will be characterized by NMR studies and X-ray crystallography. In addition, the carriers will be functionalized with a variety of fluorophores for bimodal detection. Aim #2 (year 2) is to attach targeting agents (cyclopamine, folate, glucose, somatostatin) and demonstrate specific delivery in tissue culture. In the R33 phase, Aim #3 (year 3) is to profile multiple markers simultaneously in cancer ceo lines, using xenon delivery agents optimized for specific targets. Particular efforts will be made to improve data collection procedures for in vivo applications: these include optimizing 129Xe NMR imaging parameters, protocols for delivering hyperpolarized xenon, and streamlining data analysis. MRI studies of cryptophanes in mice will be initiated, and delivery and toxicity wilt be assessed. Finally, Aim #4 (year 4) is to demonstrate the early detection of pancreatic tumors in mice using MRI. By this stage, an "arsenal" of cryptophanes and other imaging reagents will be in hand. Their deliverability, tumor selectivity, marker specificity, and 129Xe signal intensity will be evaluated, 129Xe MRI results will be validated using fluorescently labeled carriers and subsequently imaging tissue in vivo and ex vivo: such "ground truthing" will help to evaluate the diagnostic accuracy of this new technology. Overall, the R33 stage will focus on the in detection of multiple biomarkers for the early detection of pancreatic and brain cancers and potentially other diseases. [unreadable] [unreadable]